Chlamydia trachomatis (CT) infection is highly prevalent and causes significant reproductive morbidity. Despite prevention and control measures, CT infection rates are at an all-time high and reinfection is common, suggesting that protective immunity to CT is often insufficient. Control of CT infection will require a vaccine. Our long-term goal is to determine immunogenetic factors that mediate protection against CT in humans to facilitate vaccine development. We found that a CT-specific systemic CD4+ IFN-g response was associated with lower CT reinfection frequency in women. We detected IFN-g in cervicovaginal lavages but could not identify the cellular source because viable mucosal mononuclear cells (MMC) counts in lavages were too low for functional immune studies. To solve this, we analyzed menstrual blood (MB) as an MMC source, which yielded sufficient MMC counts. Preliminary data from MB MMC studies revealed IFN-g-producing tissue-resident memory T cells (Trms) as an IFN-g source, but other MMC sources of IFN-g have not been studied. Mechanisms by which IFN-g protects against CT in women remain to be elucidated, but in vitro studies suggest they may include tryptophan depletion, reactive nitrogen intermediates, glucose starvation, and cytolysis. Our preliminary data suggests that microRNAs (miRs) and genetic variants also influence CT reinfection risk. We found differential miR expression of select miRs in relation to CT reinfection. We showed in two distinct cohorts that HLA-DQB1*06 was associated with CT reinfection risk, however, its role in reinfection has not been established as neighboring HLA variants that may be in linkage disequilibrium (LD) have not been studied. The goal of this application is to bridge the gap in knowledge needed for CT vaccine development by determining IFN-g mechanisms and other effector responses that influence adaptive immunity to CT reinfection and the miRs and gene variants that affect risk for reinfection. Our central hypothesis is that protection against CT reinfection is a multifactorial process including IFN-g produced by CT-specific T cells and other effector responses, which may be regulated by miRs and genetic variants. Using samples and data from a cohort of women with vs. without CT reinfection, we now aim to further test this hypothesis through additional research: Aim 1: Evaluate mechanisms through which systemic CD4+ T cells and MMCs mediate protection against CT reinfection - IFN-g mechanisms and other effector responses from systemic CD4+ T cells and MMCs will be measured by flow-cytometry-based methods and/or PCR (Subaim 1a) and sera tested for T cell-associated miRs with a miR qPCR array (Subaim 1b); and Aim 2: Determine whether HLA-DQB1*06 or other neighboring HLA variants in LD strongly predicts CT reinfection risk - DNA will be sequenced for the HLA-DQ and -DR region and fine-mapping done to identify putative risk HLA variants for analyses with Aim 1 immune response data. This innovative research will advance CT vaccine development.